Plant-based molecular farming is an attractive opportunity for the production of recombinant proteins destined to be used in the field of human and animal health, preferably due to the potentially low production cost and attempts by biopharmaceutical industry to eliminate animal-derived proteins from manufacturing processes because of possible contamination of these products by human pathogens such as Bovine Spongiform Encephalopathy (BSE) or Creuzfeld-Jacob Disease (CJD, vCJD). However, high-yield production of hetero-oligomeric proteins in plant cells is a problem that cannot be resolved with the help of standard expression systems based on the use of strong constitutive or tissue-specific promoters for the following reasons: firstly, the majority of such recombinant proteins have a deleterious effect on plant growth and development, thus strongly compromising the yield; secondly, use of tissue-specific promoters (e.g. seed-specific) would require stable incorporation of genes encoding for pharmaceutical proteins into the genomes of edible crop plants (e.g. rice, corn, wheat), that might cause problems with transgene flow control in case of open field cultivation. Also, these systems are not commercially viable, if used in closed (greenhouse) environment due to the low yield of the product.
Plant virus-based transient expression systems (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al. 1999, Curr. Top. Microbiol. Immunol, 240, 81-94; Gleba et al., 2004, Curr. Opin. Plant Biol., 7, 182-188) are able to provide for high expression levels in plant leaf tissues and to some extent are capable to address the problems of cytotoxicity of recombinant proteins and their detrimental effect on plant development, as the technology allows to separate the growth and production stages. The best-established and commercially viable systems are based on plus-sense single-stranded RNA viral vectors, preferably on Tobacco Mosaic Virus (TMV)-derived vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al. 2002, Nature Biotechnol. 20, 622-625; U.S. Pat. No. 5,316,931; U.S. Pat. No. 5,589,367; U.S. Pat. No. 5,866,785; U.S. Pat. No. 5,977,438; WO02088369; WO02097080; WO0229068; U.S. Pat. No. 5,491,076). However these systems suffer from serious limitations that restrict their use to the production of simple, relatively small proteins. In part this is caused by the instability of viral vectors and the high frequency of their reversion to wild type, if they carry heterologous sequences larger than 1 kb. Also, serious limitation of the technology is the absence of viral vector systems capable of expressing complex hetero-oligomeric proteins like therapeutic monoclonal antibodies and their derivatives that represent the most valuable group of recombinant proteins.
There is only one publication addressing the expression of a full-length monoclonal antibody in plants using plant viral vectors (Verch et al., 1998, J. Immunol. Meth., 220, 69-75). This paper describes the use of two systemic TMV-based viral vectors for the expression of heavy and light chains of monoclonal antibody in systemic leaves, whereby the different chains are expressed from different vectors upon co-infection of N. benthamiana plants with in vitro synthesised transcripts of said vectors. However, the yield of recombinant protein in said system is so low that the presence of assembled monoclonal antibody had to be confirmed with highly sensitive tests like Western blotting and ELISA. Due to the negligible yield of recombinant antibody, this system is not suitable for practical applications and has no commercial value. Since two or more TMV-based viral vectors are normally not present in the same plant tissues of an infected plant (see example 1), the detected antigen binding activity may be due to antibody that was in vitro assembled during the isolation procedure from heavy and light antibody chains expressed in separate cells or tissue. It was previously shown that functional antibodies can be assembled in vitro from denatured and reduced antibody components (Petersen & Dorrington, 1974, J. Biol. Chem., 17, 5633-5641; Maeda et al. 1996, Protein Engineering, 9, 95-100). However, the efficiency of such assembly in the absence of conditions favourable for such an assembly is very low.
Therefore, there is no large-scale expression system for recombinant hetero-oligomeric proteins in plants, the yield and efficiency of which would be sufficient to compete on the market with other large-scale expression systems like fungal or insect cell expression systems. Such a plant expression would have to fulfil the following criteria as good as possible:    (i) high yield, including expression of the hetero-oligomeric protein of interest in as many plant tissues as possible and in as many cells of said tissues;    (ii) for preventing a deleterious effect of recombinant protein expression on plant growth, expression of the protein or product of interest should be transient (or switchable) such that expression can be started at a desired stage of plant development;    (iii) to provide for an optimal ratio of polyproteins encoding for different subunits of hetero-oligomeric protein in plant cell, thus supporting for high yield of recombinant protein at the level of said recombinant protein assembly from said subunits.
Therefore, it is an object of this invention to provide an efficient process of producing a hetero-oligomeric protein in a plant, plant part, or plant cell culture. A further object is the provision of a high-yield plant expression system capable of expressing hetero-oligomeric protein. It is another object of the invention to provide an efficient process of co-expressing more than one polypeptide of interest in the same plant cell. Further, it is an object of the invention to provide a fast and high-yield method for expressing antibodies in a plant, plant part or plant cell culture.